Textile and dyestuff industries are the major contributors to industrial pollutants containing dyes. These dyes are highly stable in light and are also resistant to microbial attack. Most of these dyes are azo dyes and under anaerobic conditions, the azo linkages are reduced to form aromatic amines and these are toxic and carcinogenic (Meyer, U. 1981. Biodegradation of synthetic organic colorants. Federation of European Microbiological Societies Symposium. 12: 371-385). Due to the importance attached to prevention of environmental pollution, environmental agencies all over the world are imposing strict regulations for mitigation of pollution from industries. The effluents from the textile industries containing fast colored dyes are a major source of concern for environmentalists since such dyes besides causing aesthetic damage to sites, are toxic and carcinogenic. Industrial effluents from textiles, paper and pulp industries and leather industries contain chromogenic substances as well as high concentrations of salts, especially chlorides and sulfates (Public Health Engineering-Design in Metric waste-water treatment by R. E. Bartlett, 1971, Applied Science Publishers Ltd., London). Remediation of such dye containing waste-waters using biological methods is termed bioremediation.
Normally, the textile dye waste-waters disposal includes physical-chemical treatment, waste minimization and biological treatment. Biological treatment includes biological pretreatment with activated sludge of textile waste-waters, and treatment in stabilization ponds (Groff, K. A. 1992, Textile waste. Water-Environment Research, 64: 725-729.). Unfortunately, waste-water treatment facilities are of ten unable to completely remove commercial dyestuff from contaminated waters and thus contribute to pollution of aquatic habitats. Some of these synthetic dyes are carcinogenic and are suggested to be responsible for tumor growth in some species of fish (see Bumpus J. A., B. J. Brock. 1988. Biodegradation of crystal violet by the white-rot fungus Phanerochaete chrysosporium. Applied and Environmental Microbiology, 54:1143-1150).
Various organisms have been tried for degradation of dyes in textile waste-waters and bioremediation.                (i) Reference is made to the Japanese patent JP 06047394 Titled: Organic dye degradation in waste-water, issued on: Feb. 22, 1994, wherein a green alga Chlorella vulgaris is used for degradation of methylene blue by irradiating a microalga fermentor to generate OH radicals which in turn help in degradation of the dye. The method has a disadvantage as it involves irradiating the fermentor containing microalga and thus becomes expensive.        (ii) Reference may be made to U.S. Pat. No. 5,091,089 dated Feb. 25, 1992 Title: Decolorization of dye-containing waste-water, wherein, living, dead, free, immobilized white-rot fungi Myrothecium or Ganoderma sp. have been employed for adsorption, dye degradation and color removal. These fungi have not been tested for their efficiency in color removal in the presence of sea salts.        (iii) Reference may be made to U.S. Pat. No. 5,755,514 dated Mar. 24, 1992 Title: Increasing biodegradability of xenobiotic azo dyes wherein, white-rot fungus Phanerochaete chrysosporium and actinomycetes Streptomyces spp are used in degradation of xenobiotic azo dyes. They are not shown to degrade azo dyes in the presence of sea salts.        (vi) Reference may be made to a publication wherein Phanerochaete chrysosporium is reported to degrade textile azo dyes very efficiently under conditions where lignin-modifying enzymes are produced by incubating cultures at 39° C., (Capalash, M. and P. Sharma. 1992. Biodegradation of textile azo-dyes by Phanerochaete chrysosporium. World Journal of Microbiology and Biotechnology. 8:309-312). However, it has not been shown to degrade synthetic dyes in the presence of sea salts. The fungal culture needs to be incubated at 39° C. for effective degradation, which may involve additional step during treatment of wastewater.        (v) A reference may be made to German patent (DD-290004, entitled ‘Microbial breakdown of xenobiotic dyes of triphenylmethane series’, issued on May 16, 1991) wherein, degradation of crystal violet and malachite green are brought about by oleophilic Gram-positive bacteria preferably Corynebacterium sp. IMET 11347 or Mycobacterium sp. IMET 11349. The disadvantage of this system is that the organisms have to be grown at 32° C. in 1% methanol and removal of bacterial inoculum from dye-containing waste-water will not be very easy.        
The fungus Flavodon flavus belonging to the class Basidiomycetes produces fertile basidiomata in medium containing alpha-cellulose and sometimes in malt extract agar medium on prolonged incubation. Most of the times the fungus is in non-sprouting form and can be recognized by crystals deposited around fungal hyphae.
Many lignin-degrading fungi can also degrade textile azo dyes but their growth and enzyme production in presence of synthetic sea water has not been demonstrated. The applicants have used a strain of Phanerochaete chrysosporium and observed that it does not grow in synthetic sea water. This implies that it cannot effect dye degradation in the presence of sea water either. Caplash et al., 1992 have also used a strain of Phanerochaete chrysosporium in their studies.
Although presence of lignin-modifying enzymes are reported in several fungal taxa belonging to the class Basidiomycetes, the applicants reported their presence in Flavodon flavus for the first time (Raghukumar, C., T. M. D'Souza, R. G. Thorn and C. A. Reddy, 1999. Lignin-modifying enzymes of Flavodon flavus, a basidiomycete isolated from a coastal marine environment. Applied Environmental Microbiology 65:2103-2111). Flavodon flavus, NIOCC isolate # 312 is a close relative of Irpex and the Polyporus-Trametes lineage of polypores (Ryvarden L. 1991. Genera of Polypores. Synopsis Fungorum 5, Fungiflora, Oslo, Norway). Cultural characteristics of the isolate #312 were studied in malt extract agar medium and on alphacel agar. Fertile basidiomata that occurred on this media were identified as Flavodon flavus on the basis of their smooth nonamyloid basidiospores, dimitic hyphal system with skeletal hyphae and simple septet generative hyphae, incrusted hymenal cystidia and poroid-hydnoid yellow fruiting bodies. Occurrence and taxonomy of this fungus is reported from Indian forests (Sen M. 1973. Cultural diagnosis of Indian Polyporaceae. 3. Genera Daedalea, Favolus, Ganoderma, Hexagonia, Irpex, Lenzites, Merulius, and Poria. Indian Forest Records (New Series) Forest Pathology, Vol. 2, No. 11, Dehra Dun, India). The applicants have isolated it from marine habitat for the first time. The applicants also shown that it grows much better in the presence of synthetic sea water. (The identification of this fungus was carried out by Dr. R. G. Thorn from Department of Botany, University of Wyoming, Laramie, Wyo. 82071-3165, U.S.A., who is also a co-author in the above mentioned publication).
Objects
The main objective of the present invention is to identify and provide novel white-rot fungus Flavodon flavus (K1) Ryv. Deposited at National Institute of Oceanography, (and at Agricultural Research Service Culture Collection (NRRL), 1815 North University Street, Peoria, Ill. 61604, U.S.A., bearing accession No. NRRL 30302), exhibiting lignin degrading properties.
Another object of the invention is to provide a process for removal of dyes using the lignin-modifying white-rot fungus, Flavodon flavus, for possible use in dye-containing waste-water and in saline soils. The said fungus can be efficiently utilized for the above-mentioned usage in fresh water as well as under estuarine conditions because of its tolerance to sea salts.
Yet another objective is to provide a process for cultivation of the novel fungus on a large scale using inexpensive raw material such as sugarcane bagasse suspended in distilled water or 50% artificial sea water or simple medium like malt extract broth prepared with fresh water as well as 50% artificial sea water.